CN111286489B

CN111286489B - Tumor angiogenesis model and preparation method and application thereof

Info

Publication number: CN111286489B
Application number: CN202010107828.2A
Authority: CN
Inventors: 王宣之; 龙小燕
Original assignee: East China Institute Of Digital Medical Engineering; First Affiliated Hospital of Wannan Medical College
Current assignee: East China Institute Of Digital Medical Engineering; First Affiliated Hospital of Wannan Medical College
Priority date: 2020-02-21
Filing date: 2020-02-21
Publication date: 2022-04-15
Anticipated expiration: 2040-02-21
Also published as: CN111286489A

Abstract

The invention provides a tumor angiogenesis model and a preparation method and application thereof. The tumor angiogenesis model comprises hydrogel microfibers in a structure of cylindrical bodies, the hydrogel microfibers having a shell structure and a core structure in contact, and the shell structure being located radially outward of the core structure; wherein the shell structure is derived from a first hydrogel material loaded with tumor cells; the core structure is derived from a second hydrogel material. The tumor angiogenesis model can simulate the three-dimensional structure of a tumor tissue microenvironment in vivo, and is beneficial to the paracrine and autocrine functions of tumor cells and endothelial cells. In addition, the two cells can not generate contact inhibition in the growth process and accord with the distribution rule of in vivo tumor cells.

Description

Tumor angiogenesis model and preparation method and application thereof

Technical Field

The invention belongs to the field of biological three-dimensional printing or tumor models, and particularly relates to a tumor angiogenesis model and a preparation method thereof.

Background

Traditional methods for studying tumor angiogenesis in vitro mainly involve culturing tumor cells and/or endothelial cells in 2D dishes, while in vivo studies mainly rely on animal models.

Firstly, 2D culture has the advantages of simple operation, controllable culture conditions, etc., but the cells cultured by 2D culture grow in a monolayer, lack of interaction between cell-cell and cell-extracellular matrix, and cannot simulate the three-dimensional structure of the in vivo tumor tissue microenvironment, and the interaction between cells and the three-dimensional microenvironment are crucial for tumor growth and angiogenesis. Second, 2D cultured cells have sufficient oxygen and nutrients that are not compatible with the presence of hypoxic environments and the concentration gradients of biological factors in solid tumors in vivo. The 2D model may therefore produce misleading results, providing erroneous guidance for future clinical trials. Furthermore, cytokines secreted by 2D cultured cells diffuse in the culture medium and do not reach effective biological concentrations, which is detrimental to the paracrine and autocrine functions of the cells, thereby showing differences from in vivo tumor cells in terms of protein expression, cell signaling, cell activity, and response to drugs.

The animal model has species difference and partial experimental animals lack immune response, so that the growth speed of the xenograft tumor is faster than that of the human tumor, immature blood vessels in the xenograft tumor cannot be compared with tumorigenic blood vessels established in a human body for a long time, the treatment effect in the human body cannot be accurately predicted by animal experimental results, and meanwhile, the animal model is not beneficial to the research of a tumor angiogenesis mechanism due to more self-interference factors and poor controllability in the animal body. In view of the limitations of 2D culture and animal models described above, more and more researchers are beginning to use 3D models to study the mechanisms of tumor angiogenesis.

The 3D culture provides a geometrical structure and a microenvironment required by in-vivo cell growth for tumor cells, promotes the interaction between the cells and extracellular matrix, and is closer to in-vivo tissues in the aspects of cell proliferation, migration, invasion, cell signaling, gene expression and the like. The 3D culture combines the advantages of 2D monolayer culture and animal in vivo experiments, not only has the advantages of intuition, condition controllability and the like of in vitro cell culture, but also can obtain biological phenotype similar to in vivo experiments.

At present, most of 3D models for studying tumor angiogenesis are to co-culture tumor cells and endothelial cells in a three-dimensional environment, and promote endothelial cell sprouting or tube formation by using vascular growth factors secreted by the tumor cells or by adding exogenous vascular growth factors. In these models, there are some drawbacks due to the presence of tumor cells and/or vascular growth factors, which are necessary to promote vascularization of endothelial cells.

Specifically, the exogenous angiogenic growth factors added into the co-cultured three-dimensional microenvironment change the inherent angiogenic growth factor concentration in the microenvironment, which is not beneficial to the paracrine and autocrine functions of tumor cells and endothelial cells. Directly mixing tumor cells and endothelial cells in a three-dimensional environment easily causes contact inhibition of the two cells in the growth process, and does not conform to the distribution rule of the tumor cells in vivo. The tumor cells are planted on the 3D model containing the endothelial cells, so that the influence of the tumor cells on the morphological structure of the endothelial cells can be better observed and analyzed, but due to artificial planting, the distribution of the tumor cells on the material is easily uneven, and the tumor cells are in a two-dimensional microenvironment, which is not favorable for the exertion of the biological functions of the tumor cells.

Therefore, how to better exert the biological function of the tumor cells in the co-culture system in the constructed 3D tumor angiogenesis model is important for researching the vascularization of the endothelial cells, because the tumor cells can participate in the angiogenesis of the tumor by directly transforming into the endothelial cells, and more importantly, the tumor cells can recruit and influence the peripheral endothelial cells to participate in the formation of new tumor vessels by secreting vascular growth factors by themselves.

Citation 1 discloses a co-culture model of microencapsulated tumor cells and endothelial cells, and specifically discloses: mixing tumor cells in 2% sodium alginate solution, spraying the cell suspension into 100mmol/L calcium chloride solution through microcapsule generator to gelatinize, washing with normal saline, reacting with 0.1% polylysine solution, washing with normal saline, reacting with 0.15% sodium alginate solution, washing with normal saline, liquefying microcapsule core with 55mmol/L sodium citrate solution, and adding CO₂Culturing in an incubator, directly using the microencapsulated tumor cells for co-culture or cryopreservation, taking endothelial cells for adherent culture or three-dimensional growth, adding the microencapsulated tumor cells into the same culture solution, co-culturing, separating the tumor cells and the endothelial cells, and performing gene and protein detection and culture solution cytokine detection.

Citation 2 discloses a tumor cell co-culture three-dimensional model, a construction method and an application thereof, and specifically discloses: the preparation method comprises the steps of preparing microspheres serving as a cell scaffold by crosslinking alginate and calcium salt, providing anchor points for cell adhesion, carrying out three-dimensional co-culture on tumor cells and fibroblasts, and carrying out cell co-culture on endothelial cells and mixed cell microspheres by a Transwell device to construct a three-dimensional co-culture tumor-blood vessel in-vitro model.

The cited documents adopt a co-culture mode to culture and construct a three-dimensional model, which is easy to cause contact inhibition of two cells in the growth process and is not in line with the distribution rule of in vivo tumor cells. The distribution of the tumor cells on the material is not uniform, and the tumor cells are in a two-dimensional microenvironment, which is not beneficial to the exertion of the biological functions of the tumor cells.

As described above, although the prior art discloses both a tumor angiogenesis model constructed by a 2D culture method and a tumor angiogenesis model constructed by a 3D culture method, the tumor angiogenesis models in the prior art have drawbacks. Therefore, it is necessary to construct a model that can exert the inherent biological properties of tumor cells to the maximum extent, and this is the key to the study of tumor angiogenesis.

Cited documents:

cited document 1: CN101157908A

Cited document 2: CN110129262A

Disclosure of Invention

Problems to be solved by the invention

Aiming at the defects existing in the tumor angiogenesis model in the prior art, the invention aims to provide the tumor angiogenesis model which can exert the inherent biological performance of tumor cells to the maximum extent.

The invention also aims to provide a preparation method of the tumor angiogenesis model, which has the advantages of easily obtained raw materials and simple and feasible preparation steps.

It is still another object of the present invention to provide a use of the aforementioned tumor angiogenesis model.

Means for solving the problems

The technical scheme related to the disclosure is as follows:

[1] a tumor angiogenesis model, wherein the tumor angiogenesis model comprises hydrogel microfibers having a structure of cylindrical bodies,

the hydrogel microfiber has a shell structure and a core structure in contact, with the shell structure being radially outward of the core structure; wherein the content of the first and second substances,

the shell structure is derived from a first hydrogel material loaded with tumor cells;

the core structure is derived from a second hydrogel material.

The tumor angiogenesis model according to [1] above, wherein the hydrogel microfibers are obtained by a bioprinting technique; the shell structure is wrapped on the radial outer side of the whole core structure; preferably, the bioprinting technique is a 3D coaxial printing technique.

The tumor angiogenesis model according to [1] or [2] above, wherein the first hydrogel material and/or the second hydrogel material comprises one or a combination of two or more of natural polymer compounds;

preferably, the natural polymer compound includes one or a combination of two or more selected from the group consisting of sodium alginate, gelatin, collagen, chitosan, and hyaluronic acid.

The tumor angiogenesis model according to any one of [1] to [3], wherein the second hydrogel material is loaded with endothelial cells;

preferably, the tumor cells and/or endothelial cells express a fluorescent protein.

[5] The preparation method of the tumor angiogenesis model comprises hydrogel microfibers which are in a cylindrical structure, wherein the preparation method of the hydrogel microfibers comprises the following steps:

the preparation method comprises the following steps: respectively preparing a first hydrogel solution and a second hydrogel solution, wherein the first hydrogel solution contains a first hydrogel material, and the first hydrogel material is loaded with tumor cells; the second hydrogel solution contains a second hydrogel material;

a printing step: and printing the first hydrogel solution into a shell structure and the second hydrogel solution into a core structure by utilizing a bioprinting technology, wherein the shell structure is in contact with the core structure, and the shell structure is positioned on the radial outer side of the core structure, so that a formed body is obtained.

[6] The method for preparing a tumor angiogenesis model according to the above [5], wherein the bioprinting technique is a 3D coaxial printing technique, and the core structure is formed at the same time as the shell structure is formed.

[7]According to the above [5]]Or [6]]The preparation method of the tumor angiogenesis model comprises the following steps of (1) preparing a first hydrogel solution, wherein the mass-to-volume ratio of the first hydrogel material in the first hydrogel solution is 10-40 mg/mL; the content of the tumor cells is 0.5 × 10⁶one/mL-10 × 10⁶Per mL; and/or

In the second hydrogel solution, the mass-to-volume ratio of the second hydrogel material is 1 mg/mL-5 mg/mL; optionally, the second hydrogel solution is loaded with endothelial cells, and the content of the endothelial cells is 0.1 × 10⁶ 1X 10 to one/mL⁶one/mL.

[8] The method for producing a tumor angiogenesis model according to [7], wherein the method further comprises a step of introducing a fluorescent protein gene into the tumor cell and/or the endothelial cell, and preferably, the fluorescent protein gene is introduced into the tumor cell and/or the endothelial cell by a virus or a liposome.

[9] The method for producing a tumor angiogenesis model according to any one of [5] to [8], wherein the method further comprises the step of crosslinking the molded body with a crosslinking agent, preferably the crosslinking agent comprises a calcium ion-containing solution; and/or

And a step of placing the molded body in an incubator to perform cultivation.

[10] The method for producing a tumor angiogenesis model according to any one of [5] to [9], wherein the method further comprises detecting a factor associated with tumor angiogenesis and/or a human-derived protein in the tumor angiogenesis model;

preferably, the factors related to tumor angiogenesis comprise one or more of VEGFA, bFGF and CD 105; and/or, the human protein comprises one or two of vWF and GFAP.

[11] Use of a tumor angiogenesis model according to any one of the above [1] to [4] or a tumor angiogenesis model prepared by the preparation method according to any one of the above [5] to [10] for the preparation of a model for in vivo and/or in vitro studies; alternatively, the in vivo and/or in vitro studies include studies of tumor angiogenesis and/or development mechanisms.

ADVANTAGEOUS EFFECTS OF INVENTION

In one embodiment of the invention, the tumor angiogenesis model of the invention can simulate the three-dimensional structure of the in vivo tumor tissue microenvironment, and is beneficial to the paracrine and autocrine functions of tumor cells and endothelial cells. In addition, the two cells can not generate contact inhibition in the growth process and accord with the distribution rule of in vivo tumor cells.

In another embodiment of the present invention, the tumor angiogenesis model of the present invention can maximize the intrinsic biological properties of tumor cells.

In another embodiment of the invention, the tumor angiogenesis model can be used for synchronously constructing a three-dimensional microenvironment for co-culture of tumor cells and endothelial cells.

In another embodiment of the invention, the tumor angiogenesis model of the invention can provide an ideal in vitro model for studying endothelial cell proliferation, chemotactic migration and lumen-like structure formation by tumor cells.

In another embodiment of the present invention, the tumor angiogenesis model of the present invention can provide an ideal in vivo model for studying the recruitment of tumor cells to host vascular endothelial cells and the involvement of tumor cells in tumor angiogenesis.

Drawings

Fig. 1 shows a schematic of a 3D coaxial printing apparatus of the invention (left) and the cross-linking step of example 1 of the invention (right).

FIG. 2 shows a photograph of a three-dimensional model of shell-U87-RFP/core-HUVEC-GFP hydrogel microfiber constructed in example 1 of the present invention under a fluorescence microscope (wherein, green: HUVEC-GFP; red: U87-RFP).

FIG. 3A is a line graph showing the secretion amount of bFGF at

days

1, 3, 5, 7, and 9 of culture for the case-U87-RFP/core-HUVEC-GFP set constructed in example 1 of the present invention, the case-U87-RFP/core set constructed in example 4, and the case/core-HUVEC-GFP set constructed in comparative example 1.

FIG. 3B is a line graph showing the secretion amounts of VEGFA at

days

FIG. 3C is a line graph showing cell proliferation of the shell-U87-RFP/core-HUVEC-GFP set constructed in example 1 of the present invention, the shell-U87-RFP/core set constructed in example 4, and the shell/core-HUVEC-GFP set constructed in comparative example 1.

FIG. 4A shows scanning electron micrographs of the microfiber three-dimensional model of shell-U87-RFP/core-HUVEC-GFP hydrogel constructed in example 1 of the present invention (day 1 of culture), U87 (black arrows) of tumor cells distributed in the shell structure, and HUVEC (white arrows) of endothelial cells distributed in the core structure.

FIG. 4B shows scanning electron micrographs of the microfiber three-dimensional model of shell-U87-RFP/core-HUVEC-GFP hydrogel constructed in example 1 of the present invention (day 7 of culture), uniformly distributed proliferating tumor cells U87 in the shell structure (black arrows), and endothelial cells HUVEC in the core structure (white arrows).

FIG. 5 shows photographs taken under a fluorescence microscope on the 7 th day of in vitro culture of a shell-U87-RFP/core-HUVEC-GFP hydrogel microfiber three-dimensional model constructed in example 1 of the present invention; wherein endothelial cells in the core structure HUVEC form lumen-like structures (green: HUVEC-GFP; red: U87-RFP).

FIG. 6 is a schematic diagram showing the number of lumen-like structures formed by endothelial cells HUVEC-GFP in the three-dimensional shell-U87-RFP/core-HUVEC-GFP hydrogel microfiber model at day 7 in vitro culture of the three-dimensional shell-U87-RFP/core-HUVEC-GFP hydrogel microfiber model constructed in example 1 of the present invention.

FIG. 7 shows photographs taken under a fluorescence microscope at day 7 of in vitro culture of the shell/core-HUVEC-GFP (without tumor cells in the shell structure) constructed in comparative example 1; among them, the endothelial cells HUVEC in the core structure did not form lumen-like structures (green: HUVEC-GFP).

FIG. 8 shows a photograph taken under a fluorescence microscope on day 7 of in vitro culture of HUVEC-GFP, endothelial cells HUVEC not forming a luminal structure, cultured with conventional 2D in comparative example 2.

FIG. 9A shows a photograph taken under a fluorescence microscope at day 7 of in vitro culture using a 3D model formed by directly mixing human glioma cells U87-RFP with human umbilical vein endothelial cells HUVEC-GFP of comparative example 3, in which no endothelial cells HUVEC forming a luminal structure (green: HUVEC-GFP; red: U87-RFP) were seen.

FIG. 9B shows a three-dimensional model of the hydrogel fiber of the planted U87-RFP/HUVEC-GFP of comparative example 4, namely: a three-dimensional model formed by planting human glioma cells U87-RFP onto a 3D model containing human umbilical vein endothelial cells HUVEC-GFP. On day 7 of in vitro culture, photographs taken under a fluorescent microscope were taken in which no HUVEC endothelial cells formed obvious luminal-like structures (green: HUVEC-GFP; red: U87-RFP).

FIG. 10 shows HE staining patterns of the three-dimensional model of shell-U87-RFP/core-HUVEC-GFP hydrogel microfibrils constructed in example 1 at day 7 of in vitro culture. Tumor cells U87 distributed in the shell structure (red arrows), endothelial cells HUVEC distributed in the core structure (black arrows).

FIG. 11 shows photographs of experimental procedures for implanting the shell-U87-RFP/core hydrogel microfiber three-dimensional model (black arrow) prepared in example 4 under the skin of nude mice in the present invention.

FIG. 12 shows a scanning electron micrograph of the shell structure of the shell-U87-RFP/core hydrogel microfiber three-dimensional model constructed in example 4 of the present invention cultured in vitro for 7 days, wherein the distribution of tumor cells U87 (black arrows) in the shell structure can be seen.

FIG. 13 shows the three-dimensional model of the shell-U87-RFP/core hydrogel microfiber constructed in example 4 of the present invention after 7 days of culture, (A) a photograph of the tumor cell U87 in the shell structure under a fluorescence microscope, (B) a photograph of the tumor cell U87 in the shell structure under a light microscope, and (C) a merged photograph of A and B.

FIG. 14 shows photographs of HE staining of xenograft tumors formed subcutaneously by implanting a shell-U87-RFP/core hydrogel microfiber three-dimensional model constructed in example 4 of the present invention into nude mice; tumor cells U87 (black arrows), first hydrogel material or second hydrogel material that was not completely absorbed (within red circles).

FIG. 15 shows immunohistochemical staining photographs of xenograft tumors formed subcutaneously by implanting a shell-U87-RFP/core hydrogel microfiber three-dimensional model constructed in example 4 of the present invention into nude mice; among these, brown stained CD105 positive endothelial cells.

FIG. 16 shows the ratio of human-derived CD 105-positive cells (denoted hCD105) and mouse-derived CD 105-positive cells (denoted mCD105) to total positive cells in xenograft tumors, respectively.

Fig. 17 shows the expression of human vWF and human GFAP in xenograft tumors. FIGS. 17 (A) - (C) double immunofluorescent staining of tumors labeled with anti-human vWF (green) and anti-human GFAP (red); nuclei were counterstained blue.

Detailed Description

Other objects, features and advantages of the present application will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

Furthermore, in the following detailed description, numerous specific details are set forth in order to provide a better understanding of the present invention. It will be understood by those skilled in the art that the present invention may be practiced without some of these specific details. In other instances, methods, means, devices and steps which are well known to those skilled in the art have not been described in detail so as not to obscure the invention.

In the present specification, the numerical range represented by "numerical value a to numerical value B" means a range including the end point numerical value A, B.

In this specification, the term "three-dimensional" has the same meaning as "3D", and three-dimensional means a spatial system formed by adding a direction vector to a planar two-dimensional system.

In this specification, the term "2D" has the same meaning as "two-dimensional" and refers to the content on one plane.

In this specification, the term "G" in "21G" and "16G" refers to the outside diameter of the print head needle tube of the nozzle of the coaxial printing apparatus, which is the same as the international G-number reference size standard for syringe needles.

In the present specification, the term "VEGFA" denotes Vascular Endothelial Growth Factor A (VEGFA), which is essential for both physiological and pathological angiogenesis.

In the present specification, the term "bFGF" means basic fibroblast growth factor (bFGF), which is a kind of vascular growth factor.

In this specification, the term "CD 105" is also known as Endoglin, and CD105 is involved in angiogenesis and is a neovascular marker.

In the present specification, the term "vWF" denotes von Willebrand Factor (vWF).

In the present specification, the term "GFAP" means a Glial Fibrillary Acidic Protein (GFAP).

In the present specification, the term "U87" means "human glioma cell line U87", which is a tumor cell.

In the present specification, the term "MCF-7" means "human breast cancer cell line MCF-7", which is a tumor cell.

In the present specification, the term "HUVEC" means "Human Umbilical Vein Endothelial Cells (HUVEC)" which is an Endothelial cell.

In the present specification, the term "HBMEC" means "Human Brain Microvascular Endothelial Cell (HBMEC)" which is an Endothelial Cell.

In the present specification, "shell-tumor cell-RFP/core-endothelial cell-GFP" means that the tumor cell carrying the RFP gene is contained in the shell structure and the endothelial cell carrying the GFP gene is contained in the core structure.

In the present specification, "shell-U87-RFP/core-HUVEC-GFP" means that U87 cells carrying the RFP gene are contained in the shell structure and HUVEC cells carrying the GFP gene are contained in the core structure, and this is referred to as U87-RFP/HUVEC-GFP.

In the present specification, "shell-U118-RFP/core-HBMEC-GFP" means that U118 cells carrying the RFP gene are contained in the shell structure, and HBMEC cells carrying the GFP gene are contained in the core structure, and these are referred to as U118-RFP/HBMEC-GFP.

In the present specification, "shell-MCF-7-RFP/core-HUVEC-GFP" means that MCF-7 cells carrying an RFP gene are contained in the shell structure and HUVEC cells carrying a GFP gene are contained in the core structure, and these cells are referred to as MCF-7-RFP/HUVEC-GFP.

In the present specification, "shell/core-endothelial cell-GFP" means that only hydrogel material is present in the shell structure and that the core structure contains endothelial cells carrying the GFP gene; "Shell/core-HUVEC-GFP" means that there is only the first hydrogel material in the shell structure and the core structure contains HUVEC cells carrying the GFP gene, which can be designated HUVEC-GFP.

In the present specification, "shell-tumor cell-RFP/core" means that tumor cells carrying the RFP gene are contained in the shell structure, and only the second hydrogel material is present in the core structure; "Shell-U87-RFP/core" means that U87 cells carrying the RFP gene are contained in the shell structure and only the second hydrogel material, which can be designated as U87-RFP, is present in the core structure.

< first aspect >

The present invention provides in < first aspect > a tumor angiogenesis model comprising hydrogel microfibers having a structure of cylindrical bodies,

the core structure is derived from a second hydrogel material.

The tumor angiogenesis model can simulate the three-dimensional structure of a tumor tissue microenvironment in vivo, can be added with no exogenous vascular growth factor, can not change the inherent vascular growth factor concentration in the microenvironment, and is beneficial to the exertion of the paracrine and autocrine functions of tumor cells and endothelial cells in the three-dimensional environment. In addition, the two cells can not generate contact inhibition in the growth process and accord with the distribution rule of in vivo tumor cells. Furthermore, the tumor cells and the endothelial cells in the tumor angiogenesis model respectively carry fluorescent proteins with different colors, which is favorable for more intuitively observing and researching the interaction and the change of biological behavior of the tumor cells and the endothelial cells in a three-dimensional co-culture environment.

Specifically, the tumor angiogenesis model of the present invention comprises the following structure:

< Shell Structure >

In the present invention, the tumor angiogenesis model mainly comprises hydrogel microfibers having a shell structure and a core structure in contact, and the shell structure is located radially outside the core structure, and preferably, the shell structure wraps the entire core structure radially outside.

In particular, the shell structure of the present invention is derived from a first hydrogel material. The first hydrogel material of the present invention may be a variety of hydrogel materials commonly used in the art. For example, the first hydrogel material of the present invention may be selected from one or a combination of two or more of natural polymer compounds. Specifically, the natural high molecular polymer may be one or a combination of two or more of sodium alginate, gelatin, collagen, chitosan, hyaluronic acid, and the like.

The first hydrogel material has good biocompatibility, hydrophilicity and degradability, has good mechanical properties after chemical crosslinking, and has a good supporting effect when being used for preparing the shell structure.

Preferably, the first hydrogel material can be selected from sodium alginate, and the sodium alginate is not only favorable for cell adhesion and growth, but also can enable the hydrogel to obtain good mechanical properties after crosslinking.

Further, the first hydrogel material of the present invention is loaded with tumor cells. The tumor cells can grow well in the first hydrogel material, and the tumor cells are in a three-dimensional microenvironment, so that the biological functions of the tumor cells can be exerted. In the present invention, the type of tumor cell is not particularly limited. By way of example, the tumor cell may be: glioma cells, glioma stem cells, breast cancer cells, lung cancer cells, liver cancer cells, cervical cancer cells and the like. Preferably, the tumor cell is a human tumor cell.

In some embodiments, the tumor cells of the invention can express a fluorescent protein. The fluorescent protein of the present invention is not particularly limited, and may be any of those commonly used in the art. For example: the fluorescent protein may be red fluorescent protein (RGF), Green Fluorescent Protein (GFP), Enhanced Green Fluorescent Protein (EGFP), Yellow Fluorescent Protein (YFP), Blue Fluorescent Protein (BFP), Cyan Fluorescent Protein (CFP), etc. Preferably, the fluorescent protein of the present invention is selected from the group consisting of Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP).

In other embodiments, after obtaining the shell structure of the present invention, the shell structure may be subjected to a cross-linking treatment, and preferably, may be subjected to a cross-linking treatment using a calcium ion-containing solution. For example: and performing crosslinking treatment by using a calcium chloride solution. The mechanical strength of the hydrogel can be enhanced through crosslinking treatment, the crosslinked shell structure can be further promoted to wrap the radial outer side of the core structure, so that an anatomical structure similar to a real vascular wall can be obtained, endothelial cells inside a lumen can be prevented from being lost, the concentration of the endothelial cells is ensured, and the interaction of the tumor cells and the endothelial cells in a three-dimensional co-culture environment can be observed and researched more intuitively.

In addition, in the present invention, the thickness of the shell structure may be 1000 μm to 2000 μm. Specifically, when the extrusion rate of the shell liquid is 15mL/h, the thickness of the shell structure is 1200 μm to 1800 μm, preferably 1400 μm to 1600 μm.

The term "crosslinking" as used herein means the same or similar meaning as "crosslinking modification", and may have some features of "modification" during the "crosslinking" process.

< core Structure >

The core structure of the present invention is located radially inward of the shell structure. In the present invention, the core structure is derived from a second hydrogel material.

The second hydrogel material of the present invention may be a variety of hydrogel materials commonly used in the art, and may be the same or different from the first hydrogel material. For example, the second hydrogel material of the present invention may also be selected from one or a combination of two or more of natural polymer compounds. The natural high molecular polymer can be one or more of sodium alginate, gelatin, collagen, chitosan, hyaluronic acid, etc.

Preferably, the second hydrogel material is selected from collagen, which is not only beneficial to the adhesion and growth of cells, but also beneficial to the formation of vascular cavity-like structures by endothelial cells.

In some specific embodiments, the second hydrogel material may be loaded with endothelial cells. When the tumor angiogenesis model contains endothelial cells, the tumor angiogenesis model can be used for synchronously constructing a three-dimensional microenvironment for co-culture of the tumor cells and the endothelial cells, so that an ideal in-vitro model is provided for further research on proliferation, chemotactic migration and lumen-like structure formation of the tumor cells on the endothelial cells.

In other specific embodiments, the second hydrogel material is not loaded with endothelial cells, and the constructed shell-tumor cell/core-hydrogel material is implanted into an animal for in vivo model construction.

The tumor cells of the invention can participate in tumor angiogenesis by directly transdifferentiating into endothelial cells, and more importantly, the tumor cells can recruit and influence peripheral endothelial cells to participate in the formation of tumor neovascularization by secreting vascular growth factors.

In addition, in the present invention, the thickness of the core structure may be 300 μm to 1000 μm. Specifically, when the extrusion rate of the core liquid is 5mL/h, the thickness of the core structure is 400 μm to 800 μm, preferably 500 μm to 700 μm.

Specifically, in the present invention, the kind of endothelial cells is not particularly limited, and may be some endothelial cells commonly used in the art. For example, the endothelial cells can be: umbilical vein endothelial cells, brain microvascular endothelial cells, arterial vascular endothelial cells, and the like. Preferably, the endothelial cells are human endothelial cells.

In some embodiments, the endothelial cells of the invention can express a fluorescent protein. The fluorescent protein of the present invention is not particularly limited, and may be any of those commonly used in the art. For example: the fluorescent protein may be red fluorescent protein (RGF), Green Fluorescent Protein (GFP), Enhanced Green Fluorescent Protein (EGFP), Yellow Fluorescent Protein (YFP), Blue Fluorescent Protein (BFP), Cyan Fluorescent Protein (CFP), etc. Preferably, the fluorescent protein may be selected from the group consisting of Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP).

In the invention, the fluorescent protein expressed by the tumor cell and the fluorescent protein expressed by the endothelial cell can be the same or different, preferably, the fluorescent protein expressed by the tumor cell and the fluorescent protein expressed by the endothelial cell are different, thereby being convenient for observing the growth conditions of the tumor cell and the endothelial cell. For example, tumor cells are made to express Red Fluorescent Protein (RFP) and endothelial cells are made to express Green Fluorescent Protein (GFP). When the light source is in the excitation wavelength range of Red Fluorescent Protein (RFP) when the fluorescence microscope is adopted for observation, the tumor cells expressing the Red Fluorescent Protein (RFP) are red; when the light source is in the excitation wavelength range of the Green Fluorescent Protein (GFP), the endothelial cells expressing the Green Fluorescent Protein (GFP) are green, so that the growth conditions of the two cells can be intuitively and conveniently observed.

Bio-printing technology

In some embodiments, the present invention utilizes bioprinting techniques to obtain the hydrogel microfibers; the shell structure may be wrapped radially outward of the entire core structure using bioprinting techniques. Preferably, the bioprinting technique is a 3D coaxial printing technique. 3D coaxial printing is also called coaxial extrusion type biological printing, and is a novel method for constructing a simulated cell three-dimensional microenvironment. Based on the diversity of coaxial channels, thread-like structures containing multiple materials and multiple cells can be simultaneously fabricated. With the help of this printing technology, the constructed cell-carrying shell-core structure has been used for the study of tissue engineering, drug screening and in vitro simulation of tissue organs. The classical "shell-core" structure consists of a shell supported by biomaterial and a core filled with cells, both the cells in the shell and the cells in the core are in a three-dimensional microenvironment, which is favorable for the exertion of the inherent biological functions of the cells.

The tumor angiogenesis model synchronously constructs a three-dimensional microenvironment for co-culture of tumor cells and endothelial cells, exerts the inherent biological properties of the tumor cells and the endothelial cells to the greatest extent, and provides an ideal in-vitro model for researching the proliferation, chemotactic migration and lumen-like structure formation of the endothelial cells by the tumor cells. Furthermore, the model provides an ideal in vivo model for researching the recruitment of tumor cells to host vascular endothelial cells and the mechanism of action of the tumor cells in tumor angiogenesis.

< second aspect >

The present invention also provides in < second aspect > a method for producing a tumor angiogenesis model according to < first aspect >, the tumor angiogenesis model including hydrogel microfibers having a structure of cylindrical bodies, wherein the method for producing the hydrogel microfibers includes the steps of:

a printing step: and printing the first hydrogel solution into a shell structure and the second hydrogel solution into a core structure by utilizing a bioprinting technology, wherein the shell structure is in contact with the core structure, and the shell structure is positioned at the radial outer side of the core structure.

Wherein the first hydrogel solution is prepared by using the first hydrogel material in the < first aspect >; the second hydrogel solution is prepared by using the second hydrogel material of < the first aspect >.

Specifically, in the first hydrogel solution, the mass-to-volume ratio of the first hydrogel material is 10 mg/mL-40 mg/mL. The first hydrogel solution may be prepared by dissolving a first hydrogel material in a sodium chloride solution. The mass concentration range of the sodium chloride solution can be 0.5-2%. Further, the first hydrogel solution contains tumor cells, and the content of the tumor cells is 0.5 x 10⁶one/mL-10 × 10⁶one/mL.

In the second hydrogel solution, the mass-to-volume ratio of the second hydrogel material is 1 mg/mL-5 mg/mL. Specifically, when the second hydrogel material is collagen, the second hydrogel solution is formulated according to the instructions of the collagen gelation procedure. The tumor angiogenesis model can be constructed according to experimental requirements, wherein the second hydrogel solution only contains the second hydrogel material without adding endothelial cells.

Specifically, the collagen gelation procedure is as follows:

1) the following items were placed on ice: collagen I, sterile 10 XPBS, sterile distilled water and sterile 1mol/L NaOH.

2) The volume of the final concentration of collagen I required for the solution to be used is determined.

3) Sterile tubes were placed on ice to store collagen I.

4) Performing the following steps under aseptic conditions:

4.1 Add 10 × PBS (final volume/10) mL;

4.2 calculate the volume of collagen I to be used (not to add to the tube until step 4.6)

Final volume Xfinal collagen I concentration (mg/mL) ═ amount of collagen to be added

Specific concentration on the bottle label (see specific lot number);

4.3 to 10 XPBS solution add (volume of collagen to be added x 0.023) mL of sterile ice-cold 1mol/L NaOH;

4.4 to the 4.3 solution was added the following volumes of sterile ice-cold distilled water:

adding distilled water volume ═ V (final) -V (collagen) -V (10 x PBS) -V (1mol/L NaOH);

4.5 mix the contents of the tube and put into ice;

4.6 Add collagen I and calculate the volume, mix well, ice for subsequent use.

5) The collagen I solution can be used immediately or left on ice for 2-3 hours.

In some specific embodiments, when the second hydrogel solution contains endothelial cells, the content of the endothelial cells is 0.1 × 10⁶ 1X 10 to one/mL⁶one/mL.

In some embodiments, the method for preparing a tumor angiogenesis model of the present invention further comprises the step of introducing a fluorescent protein gene into the tumor cells and/or the endothelial cells, such that the tumor cells and/or the endothelial cells express the fluorescent protein. The fluorescent protein according to the first aspect. In particular, the fluorescent protein gene may be introduced into the tumor cells and/or the endothelial cells by a virus or a liposome, preferably, the virus is a lentivirus. By introducing fluorescent protein genes, the growth condition of cells can be conveniently observed.

Further, with respect to the acquisition of tumor cells and/or endothelial cells, the present invention is not particularly limited, and the tumor cells and/or endothelial cells may be cultured by a conventional method until after a logarithmic growth phase, and the cells are collected for transfection experiments.

Specifically, 3X 10 can be prepared using complete medium⁴/mL～5×10⁴Perml cell suspension, 1X 10 cells per well of 24-well plate according to growth rate of each cell⁴/mL～3×10⁴Culturing the individual cells at 37 ℃ for 16-24 hours. Preferably, 2-3 μ L of 1X 10 is added to each well when the confluency of cells is 20-30%⁸TU/mL virus infection liquid for mediating RFP or GFP genes is cultured for 12-16 hours at 37 ℃, and the culture medium is replaced for continuous culture.

And (3) observing the infection efficiency by using a fluorescence microscope after 48-96 hours of infection, adding puromycin with the working concentration of 2-5 mu g/mL into the culture medium, replacing the culture solution containing puromycin every 3-4 days until the cells without virus infection are sterilized by puromycin, reducing the puromycin concentration to a maintenance concentration (1/2-1/4 of the working concentration), and continuously screening, amplifying and culturing the infected cells.

Specifically, the complete medium of the present invention may be DMEM high-glucose medium supplemented with fetal bovine serum, and the added amount of the fetal bovine serum may be 5 to 20% by mass of the total complete medium.

In some specific embodiments, the bioprinting technique is a 3D in-line printing technique such that the core structure is formed at the same time as the shell structure is formed. Therefore, the shell structure and the core structure of a tumor angiogenesis model can be synchronously constructed, and the inherent biological performance of tumor cells can be exerted to the maximum extent.

The coaxial printing technique of the present invention is realized by a coaxial printing apparatus (left part in fig. 1). The coaxial printing device comprises an outer sheath and an inner core, wherein the diameter of the outer sheath is 1.0-6.5 mm, the diameter of the inner core is 0.5-5.8 mm, and the diameters of the outer sheath and the inner core which are actually used can be selectively adjusted along with the diameter of an embedded substrate. Specifically, in printing, a shell liquid is added to the outer sheath, and a core liquid is added to the inner core. Wherein, the shell solution is a first hydrogel solution, and the core solution is a second hydrogel solution. And (3) externally connecting a micro-injection pump, slowly screwing the inner core into the outer sheath, and printing to obtain the hydrogel microfiber.

For example, the coaxial printing device mainly comprises a sheath-core coaxial nozzle which is easy to mount and dismount, and the printing head consists of a pair of 21G and 16G needle tubes. During printing, the prepared shell liquid and core liquid are respectively filled into two 5mL syringes, the head ends of the syringes are externally connected with extension tubes and are connected with a sheath channel and a core channel of a coaxial device, the syringes are placed on a micro-injection pump, and the core is slowly screwed into the sheath for printing. Specifically, the extrusion speed of the shell liquid is 5-30 mL/h, and the extrusion speed of the core liquid is 1-10 mL/h.

In some embodiments, the method for preparing a tumor angiogenesis model of the present invention further comprises the step of crosslinking the molded body with a crosslinking agent. Preferably, the cross-linking agent comprises a calcium ion-containing solution. Wherein the calcium ion-containing solution is preferably a calcium chloride solution (e.g., an aqueous calcium chloride solution); more preferably, in the calcium chloride solution, the mass-to-volume ratio of the calcium chloride is 10-50 mg/mL, and the crosslinking time is 1-5 minutes.

After crosslinking, the crosslinked molded article may be washed with a buffer such as a phosphate buffer, for example, potassium dihydrogen phosphate and/or sodium dihydrogen phosphate, and the pH of the buffer is weakly alkaline, for example, pH 7.2 to 7.5.

The mechanical strength of the hydrogel can be enhanced through crosslinking treatment, the crosslinked shell structure can be further promoted to wrap the radial outer side of the core structure, so that an anatomical structure similar to a real vascular wall can be obtained, endothelial cells inside a lumen can be prevented from being lost, the concentration of the endothelial cells is ensured, and the interaction of the tumor cells and the endothelial cells in a three-dimensional co-culture environment can be observed and researched more intuitively.

In some embodiments, the method for preparing a tumor angiogenesis model of the present invention further comprises the step of culturing the molded body in an incubator; preferably, the crosslinked molded body is cultured in an incubator.

In particular, the shaped bodies can be brought to 37 ℃ at 5% CO₂The culture medium of (4) is replaced every 3 to 4 days, and the culture medium is the complete culture medium. After cell culture, the tumor cells and the optional endothelial cells in the tumor angiogenesis model can obtain good proliferation, and the biological functions of the tumor cells can be better exerted.

In the present invention, the morphology and distribution of the tumor cells, endothelial cells and/or hydrogel material in the hydrogel microfibers may be observed using one or more of a visible light microscope, a fluorescence microscope, a scanning electron microscope, and the like.

In some embodiments of the invention, the hydrogel microfibers may be HE dyed when selected for viewing using a visible light microscope to facilitate viewing.

When the tumor cells and/or the endothelial cells carry fluorescent protein genes, the fluorescent protein can be excited by a light source in the excitation wavelength range of the fluorescent protein to generate fluorescence, so that the observation can be conveniently carried out.

In some embodiments of the invention, the sample may be dehydrated using 70%, 80%, 90%, 95%, 100% and 100% ethanol gradient, respectively, after being fixed with 2.5% glutaraldehyde at 4 ℃ overnight when observed using scanning electron microscopy, and the tumor cells in the tumor angiogenesis model of the invention and optionally the changes in morphology, structure and distribution of endothelial cells and hydrogel material may be observed by scanning electron microscopy after drying at the critical point of carbon dioxide.

In the invention, the invention can also detect the factors related to tumor angiogenesis and/or human source protein in the tumor angiogenesis model; optionally, the tumor angiogenesis-related factor comprises one or a combination of more than two of VEGFA, bFGF and CD 105; optionally, the human protein comprises one or both of vWF and GFAP.

Specifically, the method of ELISA can be used for detecting VEGFA and bFGF in the hydrogel microfiber; detecting vWF and GFAP by adopting an immunofluorescence staining method; CD105 was detected by immunohistochemical staining.

< third aspect >

The invention also provides, in a < third aspect >, an application of the tumor angiogenesis model prepared according to the < first aspect > or the preparation method of the tumor angiogenesis model of the < second aspect > in preparing a model for in vivo studies and/or in vitro studies; alternatively, the in vivo and/or in vitro studies include studies of tumor angiogenesis and/or development mechanisms.

Specifically, the tumor angiogenesis model provided by the invention can be used for synchronously constructing a co-culture three-dimensional microenvironment of tumor cells and endothelial cells, so as to analyze the influence of the shell-tumor cells on cell proliferation, chemotactic migration, luminal structure formation and angiogenesis-related marker expression of core-endothelial cells.

The tumor angiogenesis model provided by the invention can provide an ideal in-vitro model for researching proliferation, chemotactic migration and lumen-like structure formation of endothelial cells by tumor cells.

The tumor angiogenesis model provided by the invention can provide an ideal in-vivo model for researching the recruitment of tumor cells to host vascular endothelial cells and the mechanism of action of the tumor cells participating in tumor angiogenesis.

Examples

Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

In the examples, the manufacturers and models or sources of the materials and instruments used in detail are as follows:

tumor cells: human glioma cell U87, human glioma cell U118, and human breast cancer cell MCF-7 were purchased from the cell bank of Chinese academy of sciences (Shanghai, China).

Endothelial cells: HUVEC (human umbilical vein endothelial cells) and HBMEC (human brain microvascular endothelial cells) are purchased from cell banks of Chinese academy of sciences (Shanghai, China).

Complete medium (DMEM high-glucose medium + 10% fetal bovine serum):

DMEM high-glucose medium, manufacturer: gibco, cat number: c11995500 BT;

fetal bovine serum, manufacturer: gibco, cat number: 12664025.

alamar blue kit: the manufacturer: beijing Saichi Biotechnology Co., Ltd, cat #: 130012-A.

HE staining kit: the manufacturer: beijing Solaibao Tech Co., Ltd., Cat #: G1120.

OCT embedding medium: the manufacturer: shanghai Xinle Biotech, Inc., cat #: 4853.

anti-CD 105 antibodies: the manufacturer: abcam, cat # cat: ab 11414.

anti-vWF antibodies: the manufacturer: abcam, cat # cat: ab 194405.

anti-GFAP antibody: the manufacturer: abcam, cat # cat: ab 10062.

VEGFA ELISA kit: the manufacturer: donglin science and technology development, Limited liability company, Wuxi city, goods number: DL-VEGFA-Hu.

bFGF ELISA kit: the manufacturer: shanghai Yuanmu Biotech Co., Ltd., Cat No.: YM-SX 0150.

Example 1

Human glioma cell U87 and human umbilical vein endothelial cell HUVEC were cultured in complete medium respectively, and collected for transfection experiments when the two cells were in logarithmic growth phase respectively. Preparation of 3X 10 Using complete Medium⁴Perml cell suspension, 1X 10 cells per well of 24-well plate according to growth rate of each cell⁴Cells were incubated at 37 ℃ for 24 hours, and 2. mu.L of 1X 10 cells were added to each well when the confluency of cells was 30%⁸TU/mL of a viral infection solution mediating the RFP or GFP gene, wherein the viral infection solution mediating the RFP gene is added into the human glioma cells U87; adding virus infection liquid for mediating GFP gene into HUVEC of human umbilical vein endothelial cell, and culturing at 37 deg.CChanging the culture medium for further culture after 16 hours; human glioma cell U87-RFP cell suspension and human umbilical vein endothelial cell HUVEC-GFP cell suspension are respectively obtained.

And (3) observing infection efficiency by a fluorescence microscope after infection for 72 hours, adding puromycin with the working concentration of 5 mu g/mL into the culture medium, replacing the culture solution containing puromycin every 3-4 days until the cells without virus infection are killed by the puromycin, reducing the puromycin concentration to a maintenance concentration (1/2 of the working concentration), and continuously screening, amplifying and culturing the infected cells.

Weighing a certain amount of sodium alginate before printing, dissolving the sodium alginate in a sodium chloride solution with the mass concentration of 0.9%, preparing a sodium alginate solution with the mass-to-volume ratio of 20mg/mL, and sterilizing at high temperature and high pressure.

For the construction of shell-U87-RFP/core-HUVEC-GFP hydrogel microfibrils, the collected human glioma cell U87-RFP cell suspension was resuspended in a prepared sterile 20mg/mL sodium alginate solution to obtain a cell concentration of 1X 10⁶Shell liquid required for/mL coaxial printing. Preparing a collagen solution with the concentration of 2mg/mL according to the above-mentioned collagen gelation procedure instructions of the present invention, and mixing the collagen solution with the concentration of 2X 10⁵the/mL human umbilical vein endothelial cell HUVEC-GFP cell suspension is uniformly mixed with the collagen solution according to the volume ratio of 1:1 to obtain the cell concentration of 1 × 10⁵A core liquid with a collagen final concentration of 1 mg/mL.

The coaxial printing device consists of a sheath-core coaxial nozzle which is easy to mount and dismount, and the printing head consists of a pair of 21G and 16G needle tubes. During printing, the prepared shell liquid and core liquid are respectively filled into two 5mL syringes, the head ends of the syringes are externally connected with extension tubes and are connected with a shell channel and a core channel of a coaxial device, the syringes are arranged on a micro-injection pump, the extrusion speed of micro-pump shell flow is set to be 15mL/h, and the extrusion speed of core flow is set to be 5 mL/h. According to the present invention, the coaxial printing apparatus (left) shown in FIG. 1 performs coaxial bioprinting, and after the printing is completed, a molded body with cells is obtained. Using CaCl with a mass-to-volume ratio of 30mg/mL₂The petri dish of the solution served as a print-receiving platform (schematic cross-linking step (right)) to cross-link the shaped body for 2 minutesA clock.

After the coaxial bioprinting, the crosslinking agent remaining in the crosslinked molded article was washed lightly with a phosphate buffer (1X) (phosphate buffer used contains potassium dihydrogenphosphate, sodium chloride and disodium hydrogenphosphate, pH 7.2. + -. 0.1). Subsequently, the washed molded body was left at 37 ℃ with 5% CO using the complete medium described above₂The culture medium is replaced every 3 to 4 days. As shown in FIG. 2, FIG. 2 shows a photograph under a fluorescence microscope of the shell-U87-RFP/core-HUVEC-GFP hydrogel microfiber three-dimensional model constructed in example 1 of the present invention (green: HUVEC-GFP; red: U87-RFP). The shell-U87-RFP/core-HUVEC-GFP hydrogel microfiber three-dimensional model of example 1 can be referred to as the tumor angiogenesis model of the present invention.

Example 2

Human glioma cell U118 and human brain microvascular endothelial cell HBMEC are respectively cultured in a complete culture medium, and collected for transfection experiments when the two cells are respectively in logarithmic growth phase. Preparation of 4X 10 from complete Medium⁴Perml cell suspension, 2X 10 cells per well of 24-well plate according to growth rate of each cell⁴Cells were incubated at 37 ℃ for 24 hours, and 3. mu.L of 1X 10 cells were added to each well when the confluency of cells became 20%⁸TU/mL mediated RFP or GFP gene viral infection liquid, wherein, to human glioma cell U118 adding mediated RFP gene viral infection liquid; adding virus infection liquid for mediating GFP gene into human brain microvascular endothelial cell HBMEC, culturing for 14 hours at 37 ℃, and replacing culture medium for continuous culture; respectively obtaining a human glioma cell U118-RFP cell suspension and a human brain microvascular endothelial cell HBMEC-GFP cell suspension.

And (3) observing infection efficiency by a fluorescence microscope after infection for 72 hours, adding puromycin with the working concentration of 4 mug/mL into the culture medium, replacing the culture solution containing puromycin every 3-4 days until the cells without virus infection are killed by the puromycin, reducing the puromycin concentration to a maintenance concentration (1/3 of the working concentration), and continuously screening, amplifying and culturing the infected cells.

Weighing a certain amount of sodium alginate before printing, dissolving the sodium alginate in a sodium chloride solution with the mass concentration of 0.9%, preparing a sodium alginate solution with the mass-to-volume ratio of 30mg/mL, and sterilizing at high temperature and high pressure.

For the construction of shell-U118-RFP/core-HBMEC-GFP hydrogel microfibers, the collected human glioma cell U118-RFP cell suspension was resuspended in a prepared sterile 30mg/mL sodium alginate solution to obtain a cell concentration of 2X 10⁶Shell liquid required for/mL coaxial printing. Preparing collagen solution with concentration of 4mg/mL according to the above-mentioned collagen gelation procedure, mixing the collagen solution with the concentration of 4X 10⁵the/mL human brain microvascular endothelial cell HBMEC-GFP cell suspension is uniformly mixed with the collagen solution according to the volume ratio of 1:1 to obtain the cell concentration of 2 multiplied by 10⁵A core liquid with a collagen final concentration of 2 mg/mL.

The coaxial printing device consists of a sheath-core coaxial nozzle which is easy to mount and dismount, and the printing head consists of a pair of 21G and 16G needle tubes. During printing, the prepared shell liquid and core liquid are respectively filled into two 5mL syringes, the head ends of the syringes are externally connected with extension tubes and are connected with a shell channel and a core channel of a coaxial device, the syringes are arranged on a micro-injection pump, the extrusion speed of micro-pump shell flow is set to be 10mL/h, and the extrusion speed of core flow is set to be 7 mL/h. According to the present invention, the coaxial printing apparatus (left) shown in FIG. 1 performs coaxial bioprinting, and after the printing is completed, a molded body with cells is obtained. Using CaCl with a mass-to-volume ratio of 20mg/mL₂The petri dish of the solution served as a print receiving platform (cross-linking step schematic (right)) to perform cross-linking for 3 minutes.

After the coaxial bioprinting, the crosslinking agent remaining in the crosslinked molded article was washed lightly with a phosphate buffer (the phosphate buffer used contained potassium dihydrogenphosphate, sodium chloride and disodium hydrogenphosphate, pH 7.2. + -. 0.1). Subsequently, the washed molded body was left at 37 ℃ with 5% CO using the complete medium₂The culture medium is replaced every 3 to 4 days to obtain the shell-U118-RFP/core-HBMEC-GFP hydrogel microfiber three-dimensional model. The shell-U118-RFP/core-HBMEC-GFP hydrogel microfiber three-dimensional model of example 2 can be referred to as the tumor angiogenesis model of the present invention.

Example 3

Human breast cancer cell MCF-7 and human umbilical vein endothelial cell HUVEC are respectively cultured in complete culture medium, and collected for transfection experiment when the two cells are respectively in logarithmic growth phase. Preparation of 5X 10 from complete Medium⁴Perml cell suspension, 3X 10 cells per well of 24-well plate according to growth rate of each cell⁴Cells were incubated at 37 ℃ for 24 hours, and 2. mu.L of 1X 10 cells were added to each well when the confluency of cells became 20%⁸TU/mL mediated RFP or GFP gene virus infection liquid, wherein the RFP gene mediated virus infection liquid is added into human breast cancer cells MCF-7, the GFP gene mediated virus infection liquid is added into human umbilical vein endothelial cells HUVEC, the cells are cultured for 15 hours at 37 ℃, and the culture medium is replaced for continuous culture; respectively obtaining the MCF-7-RFP cell suspension of the human breast cancer cells and the HUVEC-GFP cell suspension of the human umbilical vein endothelial cells.

And (3) observing infection efficiency by a fluorescence microscope after infection for 72 hours, adding puromycin with the working concentration of 5 mu g/mL into the culture medium, replacing the culture solution containing puromycin every 3-4 days until the cells without virus infection are killed by the puromycin, reducing the puromycin concentration to a maintenance concentration (1/4 of the working concentration), and continuously screening, amplifying and culturing the infected cells.

For the construction of the shell-MCF-7-RFP/core-HUVEC-GFP hydrogel microfiber, the collected human breast cancer cell MCF-7-RFP cell suspension is resuspended in the prepared sterile 20mg/mL sodium alginate solution to obtain the cell concentration of 4X 10⁶Shell liquid required for/mL coaxial printing. Preparing collagen solution with concentration of 3mg/mL according to the above-mentioned collagen gelation procedure, mixing the collagen solution with 6X 10⁵the/mL human umbilical vein endothelial cell HUVEC-GFP cell suspension is uniformly mixed with the collagen solution according to the volume ratio of 1:1 to obtain the cell concentration of 3 multiplied by 10⁵A final collagen concentration of 1.5 mg/mL.

Coaxial printing deviceThe device consists of a sheath-core coaxial nozzle which is easy to mount and dismount, and the printing head consists of a pair of 21G and 16G needle tubes. During printing, the prepared shell liquid and core liquid are respectively filled into two 5mL syringes, the head ends of the syringes are externally connected with extension tubes and are connected with a shell channel and a core channel of a coaxial device, the syringes are arranged on a micro-injection pump, the extrusion speed of micro-pump shell flow is set to be 12mL/h, and the extrusion speed of core flow is set to be 6 mL/h. According to the present invention, the coaxial printing apparatus (left) shown in FIG. 1 performs coaxial bioprinting, and after the printing is completed, a molded body with cells is obtained. Using CaCl with a mass-to-volume ratio of 30mg/mL₂The petri dish of the solution served as a print receiving platform (cross-linking step schematic (right)) to perform cross-linking for 3 minutes.

After the coaxial bioprinting, the crosslinking agent remaining in the crosslinked molded article was washed lightly with a phosphate buffer (the phosphate buffer used contained potassium dihydrogenphosphate, sodium chloride and disodium hydrogenphosphate, pH 7.2. + -. 0.1). Subsequently, the washed molded body was left at 37 ℃ with 5% CO using the complete medium₂The culture medium is replaced every 3 to 4 days to obtain a shell-MCF-7-RFP/core-HUVEC-GFP hydrogel microfiber three-dimensional model. The three-dimensional model of the shell-MCF-7-RFP/core-HUVEC-GFP hydrogel microfiber of example 3 can be referred to as the tumor angiogenesis model of the present invention.

Example 4

Human glioma cells U87 were cultured in complete medium and collected for transfection experiments when the cells were in logarithmic growth phase. Preparation of 3X 10 Using complete Medium⁴Per mL of cell suspension, 1X 10 cells per well of 24-well plate were added according to the growth rate of the cells⁴Cells were cultured at 37 ℃ for 24 hours, and when the confluency of cells became 30%, 2. mu.L of 1X 10 cells were added to each well⁸TU/mL virus infection liquid for mediating the RFP gene is cultured for 16 hours at 37 ℃, and the culture medium is replaced for continuous culture to obtain a human glioma cell U87-RFP cell suspension.

For the construction of the shell-U87-RFP/core hydrogel microfiber, the collected human glioma cell U87-RFP cell suspension was resuspended in the prepared sterile 20mg/mL sodium alginate solution to obtain a cell concentration of 1X 10⁶Shell liquid required for/mL coaxial printing. A collagen solution (bore fluid) was prepared at a concentration of 2mg/mL according to the above-mentioned instructions for the collagen gelling procedure.

The coaxial printing device consists of a sheath-core coaxial nozzle which is easy to mount and dismount, and the printing head consists of a pair of 21G and 16G needle tubes. During printing, the prepared shell liquid and core liquid are respectively filled into two 5mL syringes, the head ends of the syringes are externally connected with extension tubes and are connected with a shell channel and a core channel of a coaxial device, the syringes are arranged on a micro-injection pump, the extrusion speed of micro-pump shell flow is set to be 15mL/h, and the extrusion speed of core flow is set to be 5 mL/h. According to the present invention, the coaxial printing apparatus (left) shown in FIG. 1 performs coaxial bioprinting, and after the printing is completed, a molded body with cells is obtained. Using CaCl with a mass-to-volume ratio of 30mg/mL₂The petri dish of solution served as the print receiving platform (cross-linking step schematic (right)) to cross-link for 2 minutes.

After the coaxial bioprinting, the crosslinking agent remaining in the crosslinked molded article was washed lightly with a phosphate buffer (the phosphate buffer used contained potassium dihydrogenphosphate, sodium chloride and disodium hydrogenphosphate, pH 7.2. + -. 0.1). Subsequently, the washed molded bodies were placed at 37 ℃ in 5% CO using the complete medium described above₂The culture medium is replaced every 3 to 4 days to obtain a shell-U87-RFP/core hydrogel microfiber three-dimensional model. The shell-U87-RFP/core hydrogel microfiber three-dimensional model of example 4 can be referred to as the tumor angiogenesis model of the present invention.

Comparative example 1

Comparative example 1 differs from example 1 only in that human glioma cell U87 was not added to the chitin solution, and then a three-dimensional shell/core-HUVEC-GFP model was constructed in exactly the same manner as in example 1.

HUVECs of human umbilical vein endothelial cells were cultured in a culture medium and collected for transfection experiments when the cells were in logarithmic growth phase. Preparation of 3X 10 Using complete Medium⁴Per mL of cell suspension, 1X 10 cells per well of 24-well plate were added according to the growth rate of the cells⁴Cells were cultured at 37 ℃ for 24 hours, and when the confluency of cells became 30%, 2. mu.L of 1X 10 cells were added to each well⁸TU/mL virus infection liquid mediating GFP gene, culturing for 16 hours at 37 ℃, and replacing culture medium for continuous culture; human umbilical vein endothelial cell HUVEC-GFP cell suspension is obtained.

For the construction of shell/core-HUVEC-GFP hydrogel microfibers, a prepared sterile 20mg/mL sodium alginate solution was used as the shell fluid required for coaxial printing. Preparing collagen solution with concentration of 2mg/mL according to the above-mentioned collagen gelation procedure, mixing 2 × 10⁵the/mL human umbilical vein endothelial cell HUVEC-GFP cell suspension is uniformly mixed with the collagen solution according to the volume ratio of 1:1 to obtain the cell concentration of 1 × 10⁵A core liquid with a collagen final concentration of 1 mg/mL.

The coaxial printing device consists of a sheath-core coaxial nozzle which is easy to mount and dismount, and the printing head consists of a pair of 21G and 16G needle tubes. Respectively mixing the prepared shell liquid and core liquid during printingThe micro-pump is arranged in two 5mL syringes, the head ends of the syringes are externally connected with extension tubes and are connected with a shell channel and a core channel of a coaxial device, the syringes are arranged on a micro-injection pump, the extrusion speed of micro-pump shell flow is set to be 15mL/h, and the extrusion speed of core flow is set to be 5 mL/h. According to the present invention, the coaxial printing apparatus (left) shown in FIG. 1 performs coaxial bioprinting, and after the printing is completed, a molded body with cells is obtained. Using CaCl with a mass-to-volume ratio of 30mg/mL₂The petri dish of solution served as the print receiving platform (cross-linking step schematic (right)) to cross-link for 2 minutes.

After the coaxial bioprinting, the residual crosslinking agent in the shell-core hydrogel microfibers was gently washed with a phosphate buffer (phosphate buffer used containing potassium dihydrogen phosphate, sodium chloride, and disodium hydrogen phosphate, pH 7.2. + -. 0.1). Subsequently, the washed molded body was left at 37 ℃ with 5% CO using the complete medium described above₂The culture medium is replaced every 3 to 4 days to obtain the shell/core-HUVEC-GFP hydrogel microfiber three-dimensional model.

Comparative example 2 (preparation of conventional 2D cultured endothelial cell-GFP)

HUVECs from human umbilical vein endothelial cells were cultured in complete medium and harvested for transfection experiments when the cells were in logarithmic growth phase. Preparation of 4X 10 from complete Medium⁴Per mL of cell suspension, 2X 10 cells per well of 24-well plate were added according to the growth rate of the cells⁴Cells were incubated at 37 ℃ for 24 hours, and 2. mu.L of 1X 10 cells were added to each well when the confluency of cells was 30%⁸TU/mL virus-infected medium mediating GFP gene was cultured at 37 ℃ for 16 hours, and the medium was replaced to continue the culture.

And (3) observing infection efficiency by a fluorescence microscope after infection for 72 hours, adding puromycin with the working concentration of 4 mu g/mL into a complete culture medium, replacing a puromycin-containing culture solution every 3-4 days until cells which are not infected with the virus are killed by the puromycin, reducing the puromycin concentration to a maintenance concentration (1/2 of the working concentration), and continuously screening, amplifying and 2D culturing the infected human umbilical vein endothelial cells HUVEC to obtain the 2D cultured human umbilical vein endothelial cells HUVEC-GFP.

Comparative example 3 (concrete preparation procedure for directly mixing tumor cell-RFP and endothelial cell-GFP to form 3D model)

Human glioma cell U87 and human umbilical vein endothelial cell HUVEC were cultured in complete medium respectively, and collected for transfection experiments when the two cells were in logarithmic growth phase respectively. Preparation of 4X 10 from complete Medium⁴Perml cell suspension, 2X 10 cells per well of 24-well plate according to growth rate of each cell⁴Cells were incubated at 37 ℃ for 24 hours, and 3. mu.L of 1X 10 cells were added to each well when the confluency of cells was 30%⁸TU/mL of viral infection solution mediating RFP or GFP gene, wherein the viral infection solution mediating RFP gene was added to human glioma cell U87, the viral infection solution mediating GFP gene was added to human umbilical vein endothelial cell HUVEC, cultured at 37 ℃ for 14 hours, and the culture medium was changed to continue the culture.

And (3) observing infection efficiency by a fluorescence microscope after infection for 72 hours, adding puromycin with the working concentration of 4 mug/mL into a complete culture medium, replacing a culture solution containing puromycin every 3-4 days until cells without virus infection are sterilized by puromycin, reducing the puromycin concentration to a maintenance concentration (1/3 of the working concentration), and continuously screening, amplifying and culturing the infected cells.

Weighing a certain amount of sodium alginate, dissolving the sodium alginate in 0.9% sodium chloride solution to prepare a sodium alginate solution with the mass-volume ratio of 20mg/mL, and sterilizing at high temperature and high pressure. For 3D model construction formed by mixing human glioma cells U87-RFP and human umbilical vein endothelial cells HUVEC-GFP, the collected human glioma cell U87-RFP cell suspension is resuspended in prepared sterile 20mg/mL sodium alginate solution, and the cell concentration is obtained to be 2 x 10⁶mL of the cell solution required for mixing. Preparing collagen solution with concentration of 2mg/mL according to the above-mentioned collagen gelation procedure, mixing the collagen solution with the concentration of 4X 10⁵the/mL human umbilical vein endothelial cell HUVEC-GFP cell suspension is uniformly mixed with the collagen solution according to the volume ratio of 1:1 to obtain the cell concentration of 2 multiplied by 10⁵The required solution was mixed at a final collagen concentration of 2 mg/mL.

The prepared medicine contains human nerveUniformly mixing sodium alginate solution of glioma cells U87-RFP and collagen solution containing human umbilical vein endothelial cells HUVEC-GFP according to the volume ratio of 3:1, and then using CaCl with the mass volume ratio of 20mg/mL₂The solution cross-linked the mixed hydrogel for 3 minutes.

After mixing and crosslinking, the crosslinking agent remaining in the 3D hydrogel model was washed gently with a phosphate buffer (the phosphate buffer used contained potassium dihydrogenphosphate, sodium chloride and disodium hydrogenphosphate, pH 7.2. + -. 0.1). Subsequently, the cell-loaded 3D hydrogel model was incubated at 37 ℃ with 5% CO₂The culture medium is replaced every 3 to 4 days to obtain a 3D model formed by mixing the human glioma cells U87-RFP and the human umbilical vein endothelial cells HUVEC-GFP.

Comparative example 4 (concrete preparation procedure for plating tumor cell-RFP onto 3D model containing endothelial cell-GFP)

Preparing a collagen solution with the concentration of 2mg/mL according to the above collagen gelation procedureWill be 4X 10⁵the/mL human umbilical vein endothelial cell HUVEC-GFP cell suspension is uniformly mixed with the collagen solution according to the volume ratio of 1:1 to obtain the cell concentration of 2 multiplied by 10⁵A solution with a final collagen concentration of 2 mg/mL. The prepared collagen solution containing the HUVEC-GFP of the human umbilical vein endothelial cells is added into a cell culture dish and is gelled for 30min at 37 ℃. Human glioma cells U87-RFP were collected and resuspended in complete medium to a cell concentration of 2X 10⁶The prepared U87-RFP cell solution is planted on the surface of HUVEC-GFP collagen gel of human umbilical vein endothelial cells by a micropipette.

The cell-loaded 3D hydrogel model was incubated at 37 ℃ with 5% CO₂The culture medium is replaced every 3 to 4 days to obtain the hydrogel fiber three-dimensional model of the planted U87-RFP/HUVEC-GFP.

In vitro experimental verification

1. Cell proliferation potency verification

To evaluate the cell proliferation capacity of the shell-tumor cell-RFP and core-endothelial cell-GFP in the three-dimensional model of the shell-core hydrogel microfiber of the present invention under the 3D hydrogel microenvironment, almar blue reagent was used to detect the proliferation change of cells on

days

1, 3, 5, 7, and 9 cultured in the incubators of the above examples or comparative examples, with cell hydrogel microfibers (specifically, the shell-U87-RFP/core-HUVEC-GFP group (denoted as U87-RFP/HUVEC-GFP) constructed in example 1, the shell-U87-RFP/core group (denoted as U87-RFP) constructed in example 4, and the shell/core-HUVEC-GFP group (denoted as HUVEC-GFP) constructed in comparative example 1) loaded in each group.

Working solutions of reagents were prepared according to alamar blue kit instructions, working solution prepared in fresh medium was added to each group of samples, incubated at 37 ℃ for 2 hours in the absence of light, the incubated detection solution was transferred to a 96-well plate at 100 μ L per well, and Optical Density (OD) values were read at wavelengths of 570nm and 630nm, and each group of OD values was normalized to day 1 for mapping and statistical analysis. Culture medium supernatants were taken on

days

1, 3, 5, 7, and 9 of the cell-loaded hydrogel microfiber culture of each group, and changes in secretion amounts of VEGFA and bFGF were measured according to the ELISA kit instructions, respectively. Among them, VEGFA and bFGF are two important proteins for promoting vascularization of endothelial cells, and the more secreted, the more easily vascularized the endothelial cells, and the experimental results are shown in fig. 3A to fig. 3C:

FIG. 3A is a line graph showing the secretion amount of bFGF at

days

FIG. 3B is a line graph showing the secretion amounts of VEGFA on

days

1, 3, 5, 7, and 9 of culture for the case-U87-RFP/core-HUVEC-GFP set constructed in example 1 of the present invention, the case-U87-RFP/core set constructed in example 4, and the case/core-HUVEC-GFP set of comparative example 1.

As can be seen from FIGS. 3A and 3B, the shell-U87-RFP/core-HUVEC-GFP group constructed in example 1 of the present invention exhibited large secretion amounts of bFGF and VEGFA, indicating that endothelial cells were easily vascularized. As can be seen from FIG. 3A, on days 5, 7, and 9, particularly on day 7 of the culture, the bFGF-secreting amounts of the shell-U87-RFP/core-HUVEC-GFP groups constructed in example 1 and the shell-U87-RFP/core groups constructed in example 4 were significantly higher than those of the shell/core-HUVEC-GFP group. As can be seen from FIG. 3B, on days 5, 7 and 9 of culture, the shell-U87-RFP/core-HUVEC-GFP set constructed in example 1 and the shell-U87-RFP/core set constructed in example 4 secreted VEGFA in significantly higher amounts than the shell/core-HUVEC-GFP set.

In addition, as can be seen from FIG. 3C, the shell-U87-RFP/core-HUVEC-GFP set constructed in example 1 of the present invention and the shell-U87-RFP/core set constructed in example 4 showed better cell proliferation (relative cell proliferation) than the shell/core-HUVEC-GFP set constructed in comparative example 1.

2. Fluorescent microscope for observing endothelial cell-GFP morphological change

Fluorescence microscope observations of the three-dimensional models of the shell-U87-RFP/core-HUVEC-GFP hydrogel microfibers constructed in example 1, the shell-U87-RFP/core hydrogel microfiber three-dimensional model constructed in example 4, the shell/core-HUVEC-GFP hydrogel microfiber three-dimensional model of comparative example 1, the endothelial cell HUVEC-GFP cultured in the conventional 2D of comparative example 2, comparative example 3 formed by directly mixing the human glioma cells U87-RFP with the human umbilical vein endothelial cells HUVEC-GFP, and the three-dimensional model obtained by seeding the human glioma cells U87-RFP onto the 3D model containing the human umbilical vein endothelial cells HUVEC-GFP of comparative example 4, cultured in the incubator of the above-described examples or comparative examples:

as shown in FIG. 5, FIG. 5 shows a photograph of a shell-U87-RFP/core-HUVEC-GFP hydrogel microfiber three-dimensional model constructed in example 1 of the present invention taken under a fluorescence microscope on the 7 th day of in vitro culture; wherein endothelial cells in the core structure HUVEC form lumen-like structures (green: HUVEC-GFP; red: U87-RFP).

As shown in fig. 13, fig. 13 is a photograph of (a) tumor cell U87 in the shell structure under a fluorescence microscope, (B) tumor cell U87 in the shell structure under a light microscope, and (C) a merged image of a and B, after 7 days in vitro culture of the shell-U87-RFP/core hydrogel microfiber three-dimensional model constructed in example 4 of the present invention.

On the 7 th day of culture, morphology change of HUVEC-GFP in endothelial cells in the three-dimensional model of the shell-U87-RFP/core-HUVEC-GFP hydrogel microfiber of example 1, presence or absence of chemotactic migration phenomenon and formation of a luminal structure were observed and recorded by an inverted fluorescence microscope. And collecting images by a laser confocal microscope. In order to quantify the formation of luminal-like structures, the tubular structures were represented by closed loops of cells, and the number of luminal-like structures formed was analyzed using Image J Image analysis software, as shown in fig. 6, from which fig. 6 it can be seen that the number of luminal-like structures formed by endothelial cells HUVEC-GFP in the shell-U87-RFP/core-HUVEC-GFP hydrogel microfiber three-dimensional model at day 7 of culture was 9.67 ± 3.52.

As shown in FIG. 7, FIG. 7 shows photographs taken under a fluorescence microscope of the three-dimensional model of shell/core-HUVEC-GFP hydrogel microfiber (without tumor cells in the shell structure) of comparative example 1, cultured in vitro at day 7; among them, the endothelial cells HUVEC in the core structure did not form lumen-like structures (green: HUVEC-GFP).

As shown in FIG. 8, FIG. 8 is a photograph of HUVEC-GFP cultured endothelial cells in conventional 2D of comparative example 2 taken under a fluorescence microscope at day 7 of in vitro culture, in which no formation of lumen-like structures by HUVEC endothelial cells was observed.

As shown in FIG. 9A, FIG. 9A shows photographs taken under a fluorescence microscope at day 7 of in vitro culture after forming a 3D model using direct mixing of U87-RFP and HUVEC-GFP, which is an endothelial cell, in comparative example 3, in which HUVEC, which is an endothelial cell, is not seen to form a luminal structure (green: HUVEC-GFP; red: U87-RFP).

As shown in FIG. 9B, FIG. 9B shows photographs taken under a fluorescence microscope of the tumor cells U87-RFP of comparative example 4 planted on a 3D model containing HUVEC-GFP as endothelial cells on day 7 of in vitro culture, in which HUVEC as endothelial cells were not seen to form a distinct lumenal structure (green: HUVEC-GFP; red: U87-RFP).

3. HE staining for observing distribution characteristics of endothelial cell HUVEC-GFP

On day 7 of the culture in the incubator of example 1 above, the three-dimensional model of the shell-U87-RFP/core-HUVEC-GFP hydrogel microfiber of example 1 was fixed with paraformaldehyde at room temperature at a mass/volume ratio of 40mg/mL for 1 hour, and then dehydrated in sucrose solutions at mass/volume ratios of 200mg/mL and 300mg/mL, respectively, until the bottom settled, and the sample was sliced in a cryomicrotome with a thickness of 6 to 8 μm after being embedded with OCT. The slide bearing the sample is placed in a constant temperature oven for proper drying to increase the adhesion of the sample to the slide. The results of observing and recording the distribution characteristics of U87-RFP and HUVEC-GFP in the shell-U87-RFP/core-HUVEC-GFP hydrogel microfiber three-dimensional model constructed in example 1 by HE staining according to the general line of the kit instructions are shown in FIG. 10. FIG. 10 shows HE staining patterns of the three-dimensional model of shell-U87-RFP/core-HUVEC-GFP hydrogel microfibrils constructed in example 1 at day 7 of in vitro culture. Tumor cells U87 distributed in the shell structure (red arrows), endothelial cells HUVEC distributed in the core structure (black arrows). As can be seen from FIG. 10, the distribution of U87-RFP and HUVEC-GFP in the microfiber three-dimensional model of the shell-U87-RFP/core-HUVEC-GFP hydrogel constructed in example 1 was uniform.

4. Scanning electron microscope observation shell-U87-RFP/core-HUVEC-GFP hydrogel microfiber three-dimensional model morphological structure and distribution change

The three-dimensional model of the shell-U87-RFP/core-HUVEC-GFP hydrogel microfiber of example 1 was fixed with glutaraldehyde at 4 ℃ in a mass-to-volume ratio of 25mg/mL overnight on days 1 and 7 of the culture in the incubator of example 1 described above, the sample was dehydrated using gradient ethanol at concentrations of 70%, 80%, 90%, 95%, 100% and 100%, respectively, and changes in the morphological structure and distribution in the three-dimensional model of the shell-U87-RFP/core-HUVEC-GFP hydrogel microfiber were observed by scanning electron microscopy after drying at the carbon dioxide critical point.

As shown in FIG. 4A, FIG. 4A is a scanning electron micrograph of a three-dimensional model of the shell-U87-RFP/core-HUVEC-GFP hydrogel microfibril constructed in example 1 (day 1 of culture). Tumor cells U87 distributed in the shell structure (black arrows), endothelial cells HUVEC distributed in the core structure (white arrows).

As shown in FIG. 4B, FIG. 4B is a scanning electron micrograph of the shell-U87-RFP/core-HUVEC-GFP hydrogel microfiber three-dimensional model constructed in example 1 (day 7 of culture); in this case, U87 (black arrows) which proliferated and distributed uniformly in the shell structure and HUVEC (white arrows) which were endothelial cells and distributed uniformly in the core structure were observed.

As shown in fig. 12, fig. 12 is a scanning electron microscope image of the shell structure of the shell-U87-RFP/core hydrogel microfiber three-dimensional model constructed in example 4 of the present invention cultured in vitro for 7 days, in which human glioma cells U87-RFP (black arrows) distributed in the shell structure can be seen, and the human glioma cells U87-RFP are uniformly distributed.

In vivo experimental verification

5 BALB/c nude mice (male, 4 weeks old) were prepared and all animal experiments were performed according to procedures approved by the animal protection and ethics committee of the Yige mountain hospital, southern Anhui medical college. A BALB/c nude mouse (30mg/kg) is anesthetized by intraperitoneal injection of a 1% sodium pentobarbital solution, after anesthesia is successful, the back skin is paved by conventional disinfection, the skin is cut under an aseptic condition, subcutaneous loose connective tissues are dissociated, as shown in figure 11, a shell-U87-RFP/core hydrogel microfiber three-dimensional model cultured in vitro on the 7 th day is implanted into the skin of the nude mouse, the skin incision is sutured by an absorbable thread after the graft is placed stably, and the incision is disinfected to wait for the nude mouse to be anesthetized and revived. Postoperative nude mice are raised in cages, and the incision of the operation is disinfected regularly.

All nude mice subcutaneous grafts were removed at 6 weeks after animal experiments, gross morphology and texture of the grafts were visually observed, the grafts were fixed with 40mg/mL paraformaldehyde at 4 ℃ overnight, dehydrated and paraffin-embedded, specimens were cut into 5 μm thick sections, and HE staining and immunohistochemical staining (CD105) were performed according to the general kit instructions, wherein the inside of the grafts was analyzed by HE staining for the presence of hydrogel, and for the disappearance of the shell-core structure and for the presence of glioma cells in the hydrogel. And judging whether the first hydrogel material and the second hydrogel material are absorbed and whether tumor cells existing in the shell-U87-RFP/core matrix are still present after the coaxial printed three-dimensional structure is transplanted in vivo for 6 weeks. Only the presence of human glioma cell U87 demonstrates successful construction of an in vivo tumor model.

As shown in fig. 14, fig. 14 is a staining diagram of a xenograft tumor HE formed by implanting a shell-U87-RFP/core hydrogel microfiber three-dimensional model constructed in example 4 of the present invention into a nude mouse subcutaneously; human glioma cells U87 (black arrows), the first hydrogel material or the second hydrogel material (within red circles) that were not completely absorbed.

Immunohistochemical staining of CD105 analyzed the grafts for the formation of new blood vessels. FIG. 15 is a photograph showing immunohistochemical staining of xenograft tumors formed subcutaneously by implanting a shell-U87-RFP/core hydrogel microfiber three-dimensional model constructed in example 4 of the present invention into nude mice; among these, brown stained CD105 positive endothelial cells.

The invention selects anti-CD 105 antibody with human species reactivity only and anti-CD 105 antibody with human and mouse species reactivity as primary antibodies respectively, performs immunohistochemical staining on the graft, searches the area with the most formed new blood vessels under low-power visual field (40 times and 100 times magnification) after staining, then photographs under 200 times visual field, calculates the number of single micro-blood vessels by using Image J Image analysis software, wherein any brown staining is adoptedThe endothelial cells or endothelial cell clusters are considered to be single countable microvessels, five different fields of view are selected to count CD105 positive cells or cell clusters, and 5 different fields of view (200-fold) can be selected from a lower (40-fold) immunohistochemical map. Microvessel density (MVD) — the number of counted microvessels/actual field of view area (0.74 mm) 200 times²) I.e., the number of microvessels counted per 200 times of the microscopic field. The proportion of human-derived CD105 positive cells and mouse-derived CD105 positive cells in the total positive cells in the graft is analyzed by using the density of the microvessels. As shown in FIG. 16, the transplant contained 25.63. + -. 4.29% of human-derived CD105 positive cells and 74.37. + -. 3.35% of mouse-derived CD105 positive cells.

For immunofluorescence staining, firstly fixing the transplant by paraformaldehyde with mass-volume ratio of 40mg/mL at 4 ℃ overnight, then sequentially placing the transplant in sucrose solution with mass-volume ratio of 200mg/mL for dewatering step by step until the transplant sinks, embedding the sample by OCT embedding medium, and then slicing the sample in a frozen microtome with the thickness of 6-8 μm. The slide glass with the specimen is placed in a constant temperature oven to be properly dried so as to increase the adhesion of the specimen and the slide glass, double immunofluorescence staining of vWF and GFAP is carried out according to the conventional kit specification, wherein the species reactivity of the anti-vWF antibody and the anti-GFAP antibody is only selected to be human, so as to further confirm and evaluate whether the new blood vessels in the graft are derived from the human tumor cells, and the result is shown in FIG. 17. FIG. 17 shows that endothelial cells co-express human GFAP and human vWF in the tumor tissue inside the graft, suggesting that a part of the neovascularization inside the graft is derived from human tumor cells, further confirming that the tumor cells can transdifferentiate into endothelial cells and play an important role in the process of tumor angiogenesis.

The tumor angiogenesis model is further prepared by utilizing a 3D coaxial printing technology, and a three-dimensional microenvironment co-cultured with endothelial cells is synchronously constructed, so that the tumor cells can influence the proliferation, chemotactic migration and lumen-like structure formation of the co-cultured endothelial cells in the tumor angiogenesis model.

Animal in vivo experiments for establishing the tumor angiogenesis model prove that tumor cells in the tumor angiogenesis model can play a role in recruiting host vascular endothelial cells and participate in angiogenesis of new tumors.

The above examples of the present invention are merely examples for clearly illustrating the present invention and are not intended to limit the embodiments of the present invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A tumor angiogenesis model, comprising hydrogel microfibers having a structure of cylindrical bodies,

the shell structure is derived from a first hydrogel material, the first hydrogel material is loaded with tumor cells, and the first hydrogel material is sodium alginate;

the core structure is derived from a second hydrogel material loaded with endothelial cells, the second hydrogel material being collagen;

the raw materials for preparing the tumor angiogenesis model comprise: a first hydrogel solution and a second hydrogel solution, wherein the first hydrogel solution contains the first hydrogel material; the second hydrogel solution contains the second hydrogel material,

in the first hydrogel solution, the mass-to-volume ratio of the first hydrogel material is 10 mg/mL-40 mg/mL; the content of the tumor cells is 0.5 × 10⁶one/mL-10 × 10⁶one/mL, in the second hydrogel solution, the mass-to-volume ratio of the second hydrogel material is 1mg/mL ∞5mg/mL, the content of the endothelial cells is 0.1 multiplied by 10⁶1X 10 to one/mL⁶one/mL.

2. The tumor angiogenesis model of claim 1, wherein the hydrogel microfibers are obtained using a bioprinting technique; the shell structure wraps the entire radial outer side of the core structure.

3. The tumor angiogenesis model of claim 2, wherein the bioprinting technique is a 3D co-axial printing technique.

4. The tumor angiogenesis model of any one of claims 1-3, wherein the tumor cells and/or endothelial cells express a fluorescent protein.

5. A method for preparing a tumor angiogenesis model according to any one of claims 1-4, wherein the tumor angiogenesis model comprises hydrogel microfibers having a cylindrical structure, and the method comprises the following steps:

the preparation method comprises the following steps: respectively preparing the first hydrogel solution and the second hydrogel solution;

6. The method of claim 5, wherein the bioprinting technique is a 3D in-line printing technique such that the core structure is formed at the same time as the shell structure.

7. The method of claim 5, further comprising the step of introducing a fluorescent protein gene into the tumor cells and/or the endothelial cells.

8. The method for preparing a tumor angiogenesis model according to claim 7, wherein a fluorescent protein gene is introduced into the tumor cells and/or the endothelial cells by a virus or a liposome.

9. The method of producing a tumor angiogenesis model according to claim 5, further comprising the step of crosslinking the molded body with a crosslinking agent; and/or

And a step of placing the molded body in an incubator to perform cultivation.

10. The method of claim 9, wherein the cross-linking agent comprises a calcium ion-containing solution.

11. The method of claim 5, further comprising detecting a factor associated with tumor angiogenesis and/or a human protein in the tumor angiogenesis model.

12. The method for preparing a tumor angiogenesis model according to claim 11, wherein the tumor angiogenesis-related factor comprises one or a combination of two or more of VEGFA, bFGF and CD 105; and/or, the human protein comprises one or two of vWF and GFAP.

13. Use of a tumor angiogenesis model according to any one of claims 1 to 4 or prepared by the preparation method of any one of claims 5 to 12 for the preparation of a model for in vitro studies.

14. Use according to claim 13, said in vitro studies comprising studies of the mechanisms of tumor angiogenesis and/or development.